The invention relates to an isolated and/or recombinant nucleic acid molecule, preferably a cloned DNA sequence element that can be incorporated into expression vectors for improving translation of a given mRNA. The invention enables the translation of mRNA in a cap-dependent and in a cap-independent manner in eukaryotic cells. The DNA sequence element comprises the so-called internal ribosomal entry site (xe2x80x9cIRESxe2x80x9d).
The PITSLRE protein kinase family is a large supergene family related to the master mitotic protein kinase, p34cdc2. PITSLRE protein kinases are encoded by the duplicated genes cell division cycle 2-like 1 (Cdc2L1) and Cdc2L2, which span approximately 140 kb on human chromosome 1p36.3 (Gururajan et al., 1998). These genes express almost identical protein kinases of 110 kDa, which contain at their C-terminal end the open reading frame of a smaller isoform of 58 kDa: p58PITSLRE. At present, the function of PITSLRE kinases remains elusive. The reason for the high number of different isoforms is also not clear. Only the p110 and p90 isoforms contain a 30-amino acid region comprised primarily of glutamic acid (83%) (Xiang et al., 1994). Shorter glutamic acid sequences can be found in all isoforms immediately following the first translational start site for the b1 isoform. Several highly basic regions, which could function as bipartite nuclear localization sequences, are found only in the p110 isoforms (Xiang et al., 1994).
The yeast two-hybrid system revealed a direct interaction between the RNA-binding protein, hRNPS1, and the p110 isoforms but not with the smaller isoforms (Loyer et al., 1998).
The wide variation in the expression patterns of the different isoforms and their levels of expression points to isoform-specific functions. While p110 isoforms are ubiquitously expressed in asynchronous cell populations, overexpression of p58 in eukaryotic cells leads to a late mitototic delay due to an apparent failure of cytokinesis. The cells are sequestered at late telophase for an extended period of time (Bunnell et al., 1990). The rate of cell growth in these cells is greatly diminished. Conversely, diminished p58 mRNA levels in CHO fibroblasts are associated with enhanced cell growth, as measured by increased rates of DNA replication (Meyerson et al., 1992). These observations suggest that p58 might participate in normal regulation of the cell cycle or cell death. Additionally, the chromosome region 1p36.3 is often deleted in neuroblastoma and many other tumors. Deletion of this chromosome region occurs late in oncogenesis and is correlated with aggressive tumor growth, suggesting that one or more tumor suppressor genes may reside here (Eipers et al., 1991). Another observation pointing to a role for PITSLRE kinases during cell cycle progression is that during early embryogenesis in Drosophila, when exponential proliferation takes place, the expression level of PITSLRE specific transcripts, encoding the p110 homologue, is maximal. At a later stage when cell proliferation is attenuated, PITSLRE expression drops dramatically (Sauer et al., 1996).
Polycistronic messengers occur frequently in prokaryotic systems. There has been a long-established misunderstanding that the polycistronic messengers did not occur in eukaryotic systems because of the presence of the so-called xe2x80x9ccapxe2x80x9d at the start of mRNA. Indeed, initiation of translation of the majority of eukaryotic cellular and viral mRNAs results from attachment of ribosomes to the m7G cap at the 5xe2x80x2-end of the mRNA followed by linear scanning to the initiation codon. However, initiation of translation of a smaller number of eukaryotic mRNAs is 5xe2x80x2-end and cap-independent and, instead, results from direct attachment of ribosomes to an internal ribosomal entry site within the 5xe2x80x2 nontranslated region (5xe2x80x2 NTR) of the mRNA.
IRES elements were first reported in picornaviral mRNAs which are naturally uncapped but nonetheless efficiently translated (Jang et al., 1988, J. Virol., 62:2636-43). Generally, IRES cannot be identified by sequence homology. Known IRES have been identified and defined functionally (Mountford and Smith, 1995, TIG, 11(5): 179-184). It appears that the conformation of the IRES sequence enables the binding on the ribosome.
It would be useful to identify a sequence element that endows any desired gene with the ability to be efficiently translated and to be translated in a cap-independent manner in particular. Moreover, it would be of great advantage to isolate a sequence element that is cell cycle regulated. Furthermore, it would be extremely useful to have IRES sequence elements with a preferably high translational efficiency to use in expression vectors as well as in gene therapy vectors in order to control mRNA translation and therefore protein synthesis.
It has been found that two isoforms, p110 and p58 of PITSLRE protein kinase, can be translated from the same PITSLRE by an internal ribosome entry process. This means that p110 and p58, two proteins with putative different functions, are translated from a single mRNA species by using two AUGs within the same reading frame. These two proteins share the 439 C-terminal amino acids that contain the kinase domain. The IRES in the polycistronic PITSLRE is the first IRES completely localized in the coding region of a cellular mRNA. Moreover, it was surprisingly found that the IRES element is cell cycle regulated. Translation of p58 occurs in the G2/M stage of the cell cycle.